Primordial germ cells (PGCs) are progenitors of all gametes. Mammalian PGCs originate from the epiblast (or embryonic ectoderm) during gastrulation. Recent studies have revealed that the extraembryonic ectoderm-derived signals play essential roles in mammalian PGC fate determination. It has also been shown that the extraembryonic ectoderm-produced bone morphogenetic protein 4 (BMP4) is absolutely required for PGC generation in the mouse. Our preliminary results indicate that the gene encoding BMP8B, another member of the BMP family, is expressed in a similar pattern as Bmp4 in the extraembryonic ectoderm and Bmp8b null mutants also have a severe defect in PGC generation. Therefore, two distantly related BMPs are required for PGC fate determination in mammals and their possible relationship in this process becomes a very intriguing biological question. Our gene expression studies with Bmp4 and Bmp8b null mutant embryos indicate that these two genes are not required for each other's expression in the extraembryonic ectoderm. Our genetic data through the generation of double heterozygotes strongly suggest that MBP4 and BMP8B form heterodimers to induce a PGC fate of the epiblast cells. In Specific Aim 1, we will further test the ability of BMP4/BMP8B heterodimers to specify a PGC fate of the epiblast by rescuing the PGC defects of Bmp4 and Bmp8b null mutant embryos. Although the extraembryonic ectoderm-produced BMPs play essential roles in PGC fate determination, it is not clear how they function to specify a PGC fate of the epiblast. They either act as autocrine factors to induce the expression of other signaling molecules that in turn specify a PGC fate of the epiblast, or they act as paracrine factors to directly instruct the epiblast cells toward a PGC fate. In Specific Aim 2, we will address the mechanism of BMP action by inactivating BMP type IA receptor (Alk3), which is absolutely required for PGC generation, specifically in the extraembryonic ectorderm using the Cre/lox recombination system. If an autocrine or indirect model is correct, a PGC defect will be observed in the resulting Alk3 mutant embryos. If a paracrine or direct model is corrent, no PGC defect is expected in these Alk3 mutants. Therefore, the successful execution of this proposal will allow us to better understand the developmental mechanisms of the mammalian PGC fate determination.